Homogeneity and secretion of a recombinant follicle stimulating hormone (FSH) in mammalian systems

ABSTRACT

A hybrid protein includes two coexpressed amino acid sequences forming a dimer. Each sequence contains the binding portion of a receptor, such as TBP1 or TBP2, or a ligand, such as IL-6, IFN-β and TPO, linked to α subunit of a natural heterodimeric scaffold. Each coexpressed sequence contains a corresponding subunit so as to form a heterodimer upon expression. Corresponding DNA molecules, expression vectors and host cells are also disclosed as are pharmaceutical compositions and a method of producing such proteins.

CROSS REFERENCE TO RELATED APPLICATION

This application is a division of application Ser. No. 09/756,186, filedJan. 9, 2001, now U.S. Pat. No. 6,663,867, which is a division ofapplication Ser. No. 08/804,166, filed Feb. 20, 1997, now U.S. Pat. No.6,193,972, which claims the benefit of U.S. Provisional Application No.60/011,936, filed Feb. 20, 1996, the entire contents of each of theabove applications being incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a hybrid protein comprising twocoexpressed amino acid sequences forming a dimer, each comprising:

a) at least one amino acid sequence selected from a homomeric receptor,a chain of a heteromeric receptor, a ligand, and fragments thereof; and

b) a subunit of a heterodimeric proteinaceous hormone or fragmentsthereof; in which (a) and (b) are bonded directly or through a peptidelinker, and, in each couple, the two subunits (b) are different andcapable of aggregating to form a dimer complex.

BACKGROUND OF THE INVENTION

Protein-protein interactions are essential to the normal physiologicalfunctions of cells and multicellular organisms. Many proteins in natureexhibit novel or optimal functions when complexed with one or more otherprotein chains. This is illustrated by various ligand-receptorcombinations that contribute to regulation of cellular activity. Certainligands, such as tumor necrosis factor α (TNFα), TNFβ, or humanchorionic gonadotropin (hCG), occur as multi-subunit complexes. Some ofthese complexes contain multiple copies of the same subunit. TNFα andTNFβ (collectively referred to hereafter as TNF) are homotrimers formedby three identical subunits (1-4). Other ligands are composed ofnon-identical subunits. For example, hCG is a heterodimer (5-7).Receptors may also occur or function as multi-chain complexes. Forexample, receptors for TNF transduce a signal after being aggregated toform dimers (8,9). Ligands to these receptors promote aggregation of twoor three receptor chains, thereby affording a mechanism of receptoractivation. For example, TNF-mediated aggregation activates TNFreceptors (10-12).

The modulation of protein-protein interactions can be a useful mechanismfor therapeutic intervention in various diseases and pathologies.Soluble binding proteins, that can interact with ligands, canpotentially sequester the ligand away from the receptor, therebyreducing the activation of that particular receptor pathway.Alternatively, sequestration of the ligand may delay its elimination ordegradation, thereby increasing its duration of effect, and perhaps itsapparent activity in vivo. In the case of TNF, soluble TNF receptorshave been primarily associated with inhibition of TNF activity (13-17).

Soluble binding proteins may be useful for treating human diseases. Forexample, soluble TNF receptors have been shown to have efficacy inanimal models of arthritis (18,19).

Since TNF has three binding sites for its receptor (10-12), anddimerization of the cell surface receptor is sufficient for bioactivity(8,9), it is likely that binding of a single soluble receptor to TNFwill leave open the possibility that this 1:3 complex of solublereceptor:TNF (trimer) can still bind and activate a pair of cell surfaceTNF receptors. To achieve an inhibitory effect, it would be expectedthat two of the receptor binding sites on the TNF trimer must beoccupied or blocked by the soluble binding protein. Alternatively, thebinding protein could block proper orientation of TNF at the cellsurface.

Generally speaking, the need was felt of synthesizing proteins thatcontain two receptor (or ligands) chains, as dimeric hybrid protein. SeeWallach et al., U.S. Pat. No. 5,478,925.

The primary strategy employed for generating dimeric or multimerichybrid proteins, containing binding domains from extracellularreceptors, has been to fuse these proteins to the constant regions of anantibody heavy chain.

This strategy led, for example, to the construction of CD4immunoadhesins (20). These are hybrid molecules consisting of the firsttwo (or all four) immunoglobulin-like domains of CD4 fused to theconstant region of antibody heavy and light chains. This strategy forcreating hybrid molecules was adapted to the receptors for TNF(10,16,21) and led to the generation of constructs with higher in vitroactivity than the monomeric soluble binding proteins.

It is widely held that the higher in vitro potency of the dimeric fusionproteins should translate into higher in vivo activity. One study doessupport this, revealing an at least 50-fold higher activity for ap75(TBP2)-Ig fusion protein in protecting mice from the consequences ofintravenous LPS injection (16).

However, despite the widespread utilization of immunoglobulin fusionproteins, this strategy has several drawbacks. One is that certainimmunoglobulin Fc domains participate in effector functions of theimmune system. These functions may be undesirable in a particulartherapeutic setting (22).

A second limitation pertains to the special cases where it is desirableto produce heteromeric fusion proteins, for example soluble analogs ofthe heteromeric IL-6 or type I interferon receptors. Although there arenumerous methods for producing bifunctional antibodies (e.g., byco-transfection or hybridoma fusions), the efficiency of synthesis isgreatly compromised by the mixture of homodimers and heterodimers thattypically results (23). Recently there have been several reportsdescribing the use of leucine zipper motifs to guide assembly ofheterodimers (24-26). This appears to be a promising approach forresearch purposes, but the non-native or intracellular sequencesemployed may not be suitable for chronic applications in the clinic dueto antigenicity. The efficiency of assembly and stability post assemblymay also be limitations.

On the other hand, in the particular case of TNF receptors, certainmodifications to the p55 TNF receptor have been found to facilitatehomodimerization and signaling in the absence of ligand (27,28). It hasbeen found that a cytoplasmic region of the receptor, termed the “deathdomain,” can act as a homodimerization motif (28,30). As an alternativeto an immunoglobulin hybrid protein, fusion of the extracellular domainof the TNF receptor to its cytoplasmic death domain could conceivablyresult in a secreted protein which can dimerize in the absence of TNF.Such fusion proteins have been disclosed and claimed in theInternational Patent Application WO 95/31544.

A third further strategy employed for generating dimers of soluble TNFreceptors has been to chemically cross-link the monomeric proteins withpolyethylene glycol (31).

SUMMARY OF THE INVENTION

An alternative for obtaining such dimeric proteins, offering someimportant advantages, is the one of the present invention and consistsin using a natural heterodimeric scaffold corresponding to a circulatingnon-immunoglobulin protein with a long half-life. A preferred example ishCG, a protein that is secreted well, has good stability, and has a longhalf-life (32-33). Given hCG's prominent role as a marker of pregnancy,many reagents have been developed to quantitate and study the protein invitro and in vivo. In addition, hCG has been extensively studied usingmutagenesis, and it is known that small deletions to the protein, suchas removal of five residues at the extreme carboxyl-terminus of the αsubunit, can effectively eliminate its biological activity whilepreserving its capability to form heterodimer (34,35). Small insertions,of up to 30 amino acids, have been shown to be tolerated at the amino-and carboxyl-termini of the α subunit (36), while fusion of the αsubunit to the carboxyl terminus of the β subunit also had little effecton heterodimer formation (37).

An analog of hCG in which an immunoglobulin Fc domain was fused to theC-terminus of hCG β subunit has also been reported; however, thisconstruct was not secreted and no effort was made to combine it with anα subunit (38).

Therefore, the main object of the present invention is a hybrid proteincomprising two coexpressed amino acid sequences forming a dimer, eachcomprising:

a) at least one amino acid sequence selected among a homomeric receptor,a chain of a heteromeric receptor, a ligand, and fragments thereof; and

b) a subunit of a heterodimeric proteinaceous hormone, or fragmentsthereof; in which (a) and (b) are bonded directly or through a peptidelinker, and in each couple the two subunits (b) are different andcapable of aggregating forming a dimer complex.

According to the present invention, the linker may be enzymaticallycleavable.

Sequence (a) is preferably selected among: the extracellular domain ofthe TNF Receptor 1 (55 kDa, also called TBP1), the extracellular domainof the TNF Receptor 2 (75 kDa, also called TBP2), or fragments thereofstill containing the ligand binding domain; the extracellular domains ofthe IL-6 receptors (also called gp80 and gp130); the extracellulardomain of the IFN α/β receptor or IFN γ receptor; a gonadotropinreceptor or its extracellular fragments; antibody light chains, orfragments thereof, optionally associated with the respective heavychains; antibody heavy chains, or fragments thereof, optionallyassociated with the respective light chains; antibody Fab domains; orligand proteins, such as cytokines, growth factors or hormones otherthan gonadotropins, specific examples of which include IL-6, IFN-β, TPO,or fragments thereof.

Sequence (b) is preferably selected among a hCG, FSH, LH, TSH, inhibinsubunit, or fragments thereof.

Modifications to the proteins, such as chemical or protease cleavage ofthe protein backbone, or chemical or enzymatic modification of certainamino acid side chains, can be used to render the components of thehybrid protein of the invention inactive. This restriction of activitymay also be accomplished through the use of recombinant DNA techniquesto alter the coding sequence for the hybrid protein in a way thatresults directly in the restriction of activity to one component, orthat renders the protein more amenable to subsequent chemical orenzymatic modification.

The above hybrid proteins will result in monofunctional, bifunctional ormultifunctional molecules, depending on the amino acid sequences (a)that are combined with (b). In each couple, (a) can be linked to theamino termini or to the carboxy termini of (b), or to both.

A monoclonal hybrid protein of the present invention can, for instance,comprise the extracellular domain of a gonadotropin receptor linked toone of the corresponding receptor-binding gonadotropin subunits.According to such an embodiment, the hybrid protein of the invention canbe a molecule in which, for example, the FSH receptor extracellulardomain is linked to FSH to increase plasma half-life and improvebiological activity.

This preparation can be employed to induce follicular maturation inassisted reproduction methods, such as ovulation induction or in vitrofertilisation, and to serve as a means to dramatically amplify thebiological activity of the hormone essential for the success of theprocess, thus reducing the requirement for both the hormone itself andthe number of injections to achieve ovulation.

The FSH receptor and the production of the extracellular domain of thehuman FSH receptor have been described respectively in WO 92/16620 andWO 96/38575.

According to a particular embodiment, the extracellular domain of theFSH receptor (ECD) can be fused in frame with a peptide linker thatcontains the thrombin recognition/cleavage site (29) and represents a“tethered” arm. The peptide linker links the extracellular domain of FSHwith a FSH subunit. This will allow for removal of the extracellulardomain of the FSH receptor by cleavage at the thrombin cleavage site asthe molecule comes in contact with thrombin in the systemic circulation.

In another embodiment, instead of the thrombin cleavage site, an enzymerecognition site for an enzyme that is found in greatest abundance inthe ovary is used. In this way, as the ECD-FSH molecule travels to theovary, it will be exposed to enzymes found in the highest concentrationsin that tissue and the ECD will be removed so that the FSH can interactwith the membrane bound receptor.

In yet another embodiment, instead of an enzyme recognition site, aflexible hinge region is cloned between ECD and FSH so that the ECD willnot be enzymatically removed from the hormone. In this way, when theECD-FSH molecule arrives at the ovary, a competition will be establishedbetween the hinge-attached ECD and the ECD of the FSH receptor found onthe ovarian cell membrane.

In a further preferred embodiment of the invention, the hybrid proteinconsists of the aggregation between a couple of aa sequences, one ofwhich contains TBP1 (or the fragments from aa 20 to aa 161 or to aa 190)as (a) and the α subunit of hCG as (b), and the other contains alwaysTBP1 (or the same fragments as above) as (a) and the β subunit of hCG,or fragments thereof, as (b). According to this embodiment, depending onthe particular sequence that is chosen as (b) (the entire β subunit ofhCG, or fragments or modifications thereof), the resulting hybridprotein will have one activity (only that of TBP1) or a combination ofactivities (that of TBP1 with that of hCG). In this latter case thehybrid protein can be used, for example, in the combined. treatment ofKaposi's sarcoma and metabolic wasting in AIDS.

In a further embodiment of the invention, one or more covalent bondsbetween the two subunits (b) are added to enhance the stability of theresulting hybrid protein. This can be done, e.g., by adding one or morenon-native interchain disulfide bonds. The sites for these cross-linkscan be deduced from the known structures of the heterodimeric hormones.For example, a suitable site in hCG could be to place cysteine residuesat α subunit residue Lys45 and β subunit residue Glu21, replacing a saltbridge (non-covalent bond) with a disufide bond (covalent bond). Anotherobject of the present invention are PEGylated or other chemicallymodified forms of the hybrid proteins.

A further object of the present invention is a DNA molecule comprisingthe DNA sequence coding for the above hybrid protein, as well asnucleotide sequences substantially the same. “Nucleotide sequencessubstantially the same” includes all other nucleic acid sequences which,by virtue of the degeneracy of the genetic code, also code for the givenamino acid sequence.

For the production of the hybrid protein of the invention, the DNAsequence (a) is obtained from existing clones, as is (b). The DNAsequence coding for the desired sequence (a) is ligated with the DNAsequence coding for the desired sequence (b). Two of these fusedproducts are inserted and ligated into a suitable plasmid or each into adifferent plasmid. Once formed, the expression vector, or the twoexpression vectors, is introduced into a suitable host cell, which thenexpresses the vector(s) to yield the hybrid protein of the invention asdefined above.

The preferred method for preparing the hybrid of the invention is by wayof PCR technology using oligonucleotides specific for the desiredsequences to be copied from the clones encoding sequences (a) and (b).

Expression of any of the recombinant proteins of the invention asmentioned herein can be effected in eukaryotic cells (e.g., yeasts,insect or mammalian cells) or prokaryotic cells, using the appropriateexpression vectors. Any method known in the art can be employed.

For example the DNA molecules coding for the proteins obtained by any ofthe above methods are inserted into appropriately constructed expressionvectors by techniques well known in the art (see Sambrook et al, 1989).Double stranded cDNA is linked to plasmid vectors by homopolymerictailing or by restriction linking involving the use of synthetic DNAlinkers or blunt-ended ligation techniques: DNA ligases are used toligate the DNA molecules and undesirable joining is avoided by treatmentwith alkaline phosphatase.

In order to be capable of expressing the desired protein, an expressionvector should comprise also specific nucleotide sequences containingtranscriptional and translational regulatory information linked to theDNA coding the desired protein in such a way as to permit geneexpression and production of the protein. First in order for the gene tobe transcribed, it must be preceded by a promoter recognizable by RNApolymerase, to which the polymerase binds and thus initiates thetranscription process. There are a variety of such promoters in use,which work with different efficiencies (strong and weak promoters).

For eukaryotic hosts, different transcriptional and translationalregulatory sequences may be employed, depending on the nature of thehost. They may be derived form viral sources, such as adenovirus, bovinepapilloma virus, Simian virus or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Examples are the TK promoter of the Herpes virus, the SV40early promoter, the yeast gal4 gene promoter, etc. Transcriptionalinitiation regulatory signals may be selected which allow for repressionand activation, so that expression of the genes can be modulated.

The DNA molecule comprising the nucleotide sequence coding for thehybrid protein of the invention is inserted into a vector(s), having theoperably linked transcriptional and translational regulatory signals,which is capable of integrating the desired gene sequences into the hostcell. The cells which have been stably transformed by the introduced DNAcan be selected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markermay also provide for phototrophy to a auxotropic host, biocideresistance, e.g., antibiotics, or heavy metals such as copper, or thelike. The selectable marker gene can either be directly linked to theDNA gene sequences to be expressed, or introduced into the same cell byco-transfection. Additional elements may also be needed for optimalsynthesis of proteins of the invention.

Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Once the vector(s) or DNA sequence containing the construct(s) has beenprepared for expression, the DNA construct(s) may be introduced into anappropriate host cell by any of a variety of suitable means:transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,etc.

Host cells may be either prokaryotic or eukaryotic. Preferred areeukaryotic hosts, e.g., mammalian cells, such as human, monkey, mouse,and Chinese hamster ovary (CHO) cells, because they providepost-translational modifications to protein molecules, including correctfolding or glycosylation at correct sites. Also, yeast cells can carryout post-translational peptide modifications including glycosylation. Anumber of recombinant DNA strategies exist which utilize strong promotersequences and high copy number of plasmids which can be utilized forproduction of the desired proteins in yeast. Yeast recognizes leadersequences on cloned mammalian gene products and secretes peptidesbearing leader sequences (i.e., pre-peptides).

After the introduction of the vector(s), the host cells are grown in aselective medium, which selects for the growth of vector-containingcells. Expression of the cloned gene sequence(s) results in theproduction of the desired proteins.

Purification of the recombinant proteins is carried out by any one ofthe methods known for this purpose, i.e., any conventional procedureinvolving extraction, precipitation, chromatography, electrophoresis, orthe like. A further purification procedure that may be used inpreference for purifying the protein of the invention is affinitychromatography using monoclonal antibodies which bind the target proteinand which are produced and immobilized on a gel matrix contained withina column. Impure preparations containing the recombinant protein arepassed through the column. The protein will be bound to the column bythe specific antibody while the impurities will pass through. Afterwashing, the protein is eluted from the gel by a change in pH or ionicstrength.

The term “hybrid protein”, as used herein, generically refers to aprotein which contains two or more different proteins or fragmentsthereof.

As used herein, “fusion protein” refers to a hybrid protein, whichconsists of two or more proteins, or fragments thereof, linked togethercovalently.

The term “aggregation”, as used herein, means the formation of strongspecific non-covalent interactions between two polypeptide chainsforming a complex, such as those existing between the α and β subunit ofa heterodimeric hormone (such as FSH, LH, hCG or TSH).

The terms “ligand” or “ligand protein”, as used herein, refer to amolecule, other than an antibody or an immunoglobulin, capable of beingbound by the ligand-binding domain of a receptor; such molecule mayoccur in nature, or may be chemically modified or chemicallysynthesised.

The term “ligand-binding domain”, as used herein, refers to a portion ofthe receptor that is involved in binding a ligand and is generally aportion or essentially all of the extracellular domain.

The term “receptor”, as used herein, refers to a membrane protein, whosebinding with the respective ligand triggers secondary cellular responsesthat result in the activation or inhibition of intracellular process.

In a further aspect, the present invention provides the use of thehybrid protein as a medicament. The medicament is preferably presentedin the form of a pharmaceutical composition comprising the protein ofthe invention together with one or more pharmaceutically acceptablecarriers and/or excipients. Such pharmaceutical compositions representyet a further aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood by reference to the appendeddrawings, in which:

FIGS. 1( a) and 1(b) show the TBP(20-161)-hCGα and TBP(20-161)-hCGβconstructs, respectively, and the corresponding sequences (SEQ IDNOS:1-4).

FIGS. 2( a) and 2(b) show the TBP(20-190)-hCGα and TBP(20-190)-hCGβconstructs, respectively, and the corresponding sequences (SEQ IDNOS:5-8).

FIG. 3 is a schematic summary of the constructs of FIGS. 1 and 2 showingp55 TNFR1, TBP1 and TBP1 fusion constructs. The linker sequences shownon the last two lines are SEQ ID NO:9 (Ala-Gly-Ala-Ala-Pro-Gly) and SEQID NO:10 (Ala-Gly-Ala-Gly).

FIG. 4 is a graph illustrating the dose dependent protective effect ofCHO cell expressed TBP-hCG(20-190) on TNFα-induced cytotoxicity on BT-20cells and various controls.

FIG. 5 is a graph illustrating the dose dependent protective effect ofCOS cell expressed TBP-hCG(20-190) on TNFα-induced cytotoxicity on BT-20cells and various controls.

FIG. 6 is a graph illustrating the dose dependent protective effect ofaffinity purified CHO cell expressed TBP-hCG(20-161) on TNFα-inducedcytotoxicity on BT-20 cells and various controls.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention will now be described by means of the following Examples,which should not be construed as in any way limiting the presentinvention.

EXAMPLES Materials and Methods

Cell lines used in this study were obtained from the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209, unless otherwise specified. The CHO-DUKX cell line wasobtained from L. Chasin at Columbia University through D. Houseman atMIT (39). The CHO-DUKX cells, which lack a functional gene fordihydrofolate reductase, were routinely maintained in complete α-plusModified Eagles Medium (α(+)MEM) supplemented with 10% fetal bovineserum (FBS). The COS-7 cells were routinely maintained in Dulbecco'sModified Eagles Medium (DMEM) supplemented with 10% FBS. Unlessspecified otherwise, cells were split to maintain them in log phase ofgrowth, and culture reagents were obtained from GIBCO (Grand Island,N.Y.).

1. Assembly of the Genetic Constructs Encoding the Hybrid Proteins

The numbering assignments for the p55 TNF receptor are based on thecloning paper from Wallach (40), while the numbering assignments for thehCG subunits are based on the numbering assignments from the Fiddescloning papers (41,42). The designation TBP, or TNF binding protein,refers to the extracellular domain portions of the TNF receptors capableof binding TNF. In these Examples, the DNA constructs will be named asTBP-hybrid proteins, with the partner and region of TBP indicated in theconstruct nomenclature. All of the TBP-hCG constructs contain the humangrowth hormone (hGH) signal peptide in place of the native p55 signalsequence. In addition, the hGH signal peptide has been placed so that itimmediately precedes TBP residue Asp20, which is anticipated to makethis the first residue in the mature, secreted protein. Thesemodifications are not essential to the basic concept of using hCG as apartner of the hybrid protein.

The DNAs encoding the hybrid proteins were constructed using PCRmethodology (43).

a. TBP1(20-161)-hCG

The initial TBP-hCG construct was engineered to contain the ligandbinding domain from the extracellular region of the p55 TNF receptor(from Asp20 inclusive of residue Cys161) fused though a short linker tothe hCG α and β subunits (starting at residues αCys7 or βPro7,respectively). This construct, hereafter referred to asTBP1(20-161)-hCG, is a heterodimer of two modified hCG subunits,TBP1(20-161)-hCGα and TBP1(20-161)-hCGβ.

The oligodeoxynucleotide primers used for the TBP1(20-161)-hCGαconstruct were:

primer 1(αβ) TTT TCT CGA GAT GGC (SEQ ID NO:11) TAC AGG TAA GCG CCCprimer 2(α) ACC TGG GGC AGC ACC (SEQ ID NO:12) GGC ACA GGA GAC ACA CTCGTT TTC primer 3(α) TGT GCC GGT GCT GCC (SEQ ID NO:13) CCA GGT TGC CCAGAA TGC ACG CTA CAG primer 4(α) TTT TGG ATC CTT AAG (SEQ ID NO:14) ATTTGT GAT AAT AAC AAG TAC

These and all of the other primers described in these Examples weresynthesized on an Applied Biosystems Model 392 DNA synthesis machine(ABI, Foster City, Calif.), using phosphoramidite chemistry.

Since both of the TBP-hCG subunit constructs have the same 5′-end (i.e.,the 5′-end of the hGH/TBP construct), primer 1(αβ) was used for bothTBP-hCG subunit constructs. The other primers used for theTBP1(20-161)-hCGβ construct were:

primer 2(β) CCG TGG ACC AGC ACC (SEQ ID NO:15) AGC ACA GGA GAC ACA CTCGTT TTC primer 3(β) TGT GCT GGT GCT GGT (SEQ ID NO:16) CCA CGG TGC CGCCCC ATC AAT primer 4(β) TTT TGG ATC CTT ATT (SEQ ID NO:17) GTG GGA GGATCG GGG TG

Primers 2(α) and 3(α) are reverse complements, and cover both the 3′-endof the coding region for the p55 extracellular domain, and the 5′-end ofthe hCG α subunit. Similarly, primers 2(β) and 3(β) are also reversecomplements, and cover both the 3′-end of the coding region for the p55extracellular domain, and the 5′-end of the hCG β subunit.

Two PCR reactions were run for each of the two TBP-hCG subunitconstructs. The first used primers 1(αβ) and 2 (α or β), and used as thetemplate a plasmid encoding soluble p55 residues 20-180 preceded by thehGH signal peptide (plasmid pCMVhGHspcDNA.pA4). The second used primers3 (α or β) and 4 (α or β), and used as the template either plasmidpSVL-hCGα or pSVL-hCGβ (44). The PCR was performed using Vent (TM)polymerase from New England Biolabs (Beverly, Mass.) in accordance withthe manufacturer's recommendations, using for each reaction 25 cyclesand the following conditions:

100 μg of template DNA

1 μg of each primer

2U of Vent(TM) polymerase (New England Biolabs)

denaturation at 99° C. for 30 seconds

annealing at:

-   -   59° C. for 30 seconds for primers 1(αβ) and 2(α)    -   59° C. for 30 seconds for primers 3(α) and 4(α)    -   57° C. for 30 seconds for primers 1(αβ) and 2(β)    -   63° C. for 30 seconds for primers 3(β) and 4(β)    -   extension at 75° C. for 75 seconds.

The PCR products were confirmed to be the expected size byelectrophoresis in a 2% agarose gel and ethidium bromide staining. Thefragments were then purified by passage over a Wizard column (Promega)in accordance with the column manufacturer's recommendations.

The final coding sequence for TBP1(20-161)-hCGα was assembled by fusionPCR using primer 1(αβ) and primer 4(α), and using as template thepurified products from the p55 and hCG α fragments obtained from thefirst PCR reactions. First the two templates, which due to the overlapbetween primers 2(α) and 3(α) could be denatured and annealed together,were passed through 10 cycles of PCR in the absence of any addedprimers. The conditions for these cycles were essentially the same asthose used earlier, except that the annealing was done at 67° C. and theextension was performed for 2 minutes. At the end of these 10 cycles,primers 1(αβ) and 4(α) were added, and another 10 cycles were performed.The conditions for this final set of reactions was the same as usedearlier, except that an annealing temperature of 59° C. was used, andthe extension was performed for 75 seconds.

Analysis of the products of this reaction by electrophoresis in a 1%agarose gel confirmed that the expected fragment of about 1100 bp wasobtained. The reaction was passed over a Wizard column to purify thefragment, which was then digested with XbaI and BamHI and re-purified ina 0.7% low-melting point agarose gel. The purified fragment wassubcloned into plasmid pSVL (Pharmacia), which had first been digestedwith XbaI and BamHI and gel purified on a 0.8% low-melting point agarosegel. Following ligation with T4 ligase, the mixture was used totransform AG1 E. coli and then plated onto LB/ampicillin plates forovernight culture at 37° C. Plasmid DNAs from ampicillin-resistantcolonies were analyzed by digestion with XhoI and BamHI to confirm thepresence of the insert (which is excised in this digest). Six cloneswere found to contain inserts, and one (clone 7) was selected forfurther advancement and designated pSVLTBPhCGα (containingTBP1(20-161)-hCGα). Dideoxy DNA sequencing (using Sequenase™, U.S.Biochemicals, Cleveland, Ohio) of the insert in this vector confirmedthat the construct was correct, and that no undesired changes had beenintroduced.

The final coding sequence for TBP1(20-161)-hCGβ was assembled in amanner similar to that described for TBP1(20-161)-hCGα using fusion PCRand primers 1(αβ) and 4(β), and using as template the purified productsfrom the p55 and hCG β fragments obtained from the first PCR reactions.The resulting PSVL plasmid containing the insert of interest wasdesignated pSVLTBPhCGβ.

b. TBP(20-190)-hCG

A second set of TBP-hCG proteins was prepared by modification of theTBP(20-161)-hCG constructs to produce an analog containing TBP spanningfrom Asp20 to Thr190, in place of the 20-161 region in the initialanalog. This was done by replacing the fragment between the BglII andXbaI sites in plasmid pSVLTBPhCGα with a PCR fragment containing thechange. This PCR fragment was generated using fusion PCR. The primerswere:

primer 1 TTT TAG ATC TCT TCT TGC ACA (SEQ ID NO:18) GTG GAC primer 2 TGTGGT GCC TGA GTC CTC AGT (SEQ ID NO:19) primer 3 ACT GAG GAC TCA GGC ACCACA (SEQ ID NO:20) GCC GGT GCT GCC CCA GGT TG primer 4 TTT TTC TAG AGAAGC AGC AGC (SEQ ID NO:21) AGC CCA TG

Primers 1 and 2 were used to generate the sequence coding the additionalp55 residues from 161-190. The PCR reaction was performed essentially asdescribed earlier, using 1 μg of each primer and pUC-p55 as template.Similarly, primers 3 and 4 were used to generate by PCR the linkerbetween the 3′-end of the TBP-coding region, and the 5′-end of the hCG αsubunit coding region, using as a template plasmid pSVLTBPhCGα. Productsfrom these PCR reactions were confirmed to be the correct size (about296 bp and 121 bp respectively) by polyacrylamide gel electrophoresis(PAGE) on an 8% gel, and were then purified using a Wizard column. Thedesign of primers 2 and 3 was such that they contained a region ofoverlap, so that the two PCR products (from primers 1 and 2, and fromprimers 3 and 4) could be annealed for fusion PCR with primers 1 and 4.Subsequent to the fusion reaction, the desired product of about 400 bpwas confirmed and purified using a 1.5% agarose gel and a Wizard column.This DNA was then digested with BglII and XbaI, and ligated withBglII/XbaI-digested pSVLTBPhCGα. The presence of an insert in plasmidsisolated from transformed AG1 E. coli was confirmed by digestion withBglII and XbaI. The new construct was designated pSVLTBP(20-190)-hCGα.

Similarly, plasmid pSVLTBPhCGβ was modified by substitution of theBglII-XcmI fragment. However, this was done by subcloning of a singlePCR product, rather than with a fusion PCR product. Primers 1 and 2b(see below) were used with pUC-p55 as the template.

primer 2b TTT TCC ACA GCC AGG GTG (SEQ ID NO:22) GCA TTG ATG GGG CGG CACCGT GGA CCA GCA CCA GCT GTG GTG CCT GAG TCC TCA GTG

The resulting PCR product (about 337 bp) was confirmed and purified asdescribed above, digested with BglII and XcmI, and then ligated intoBglII/XbaI-digested pSVLTBPhCGβ. The presence of an insert in plasmidsisolated from transformed AG1 E. coli was confirmed by digestion withBglII and XcmI. The new construct was designated pSVLTBP(20-190)-hCGβ.

The new constructs were subsequently confirmed by DNA sequencing.

In addition to producing these new pSVL-based plasmids, these constructswere also subcloned into other expression vectors likely to be moresuitable for stable expression in CHO, particularly vector Dα,previously described as plasmid CLH3AXSV2DHFR (45). This wasaccomplished by converting a BamHI site flanking the inserts in thepSVL-based vectors to an XhoI site, and then excising the insert withXhoI and cloning it into XhoI digested Dα.

2. Transient and Stable Expression of the Hybrid Proteins

Transfections of COS-7 cells (ATCC CRL 1651, ref. 46) for transientexpression of the TBP-hCG hybrid proteins were performed usingelectroporation (47). Exponentially growing COS-7 cells were removed bytrypsinization, collected by gentle centrifugation (800 rpm, 4 minutes),washed with cold phosphate buffered saline (PBS), pH 7.3-7.4, and thenrepelleted by centrifugation. Cells were resuspended at a concentrationof 5×10⁶ cells per 400 μl cold PBS and mixed with 10 μg of plasmid DNAin a prechilled 2 mm gap electroporation cuvette. For cotransfections, 5μg of each plasmid were used. The cuvette and cells were chilled on icefor a further 10 minutes, and then subjected to electroporation using aBTX Model 600 instrument and conditions of 125 V, 950 μF and R=8.Afterward the cells were set to cool on ice for 10 minutes, transferredto a 15 ml conical tube containing 9.5 ml complete medium (Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum(FBS) and 1% L-glutamine) at room temperature, and left at roomtemperature for 5 minutes. After gentle mixing in the 15 ml tube, theentire contents was seeded onto two P100 plates and placed into a 37°C., 5% CO₂ incubator. After 18 hours the media was changed, and in somecases the new media contained only 1% or 0% FBS. After another 72 hours,the conditioned media was harvested, centrifuged to remove cells, andthen stored frozen at −70° C.

Transfections of CHO-DUKX (CHO) cells for transient or stable expressionwere performed using calcium phosphate precipitation of DNA. Twenty-fourhours prior to the transfection, exponentially growing CHO cells wereplated onto 100 mm culture plates at a density of 7.5×10⁵ cells perplate. On the day of the transfection, 10 μg of plasmid DNA was broughtto 0.5 ml in transfection buffer (see below), 31 μl of 2 M CaCl₂ wereadded, the DNA-CaCl₂ solution was mixed by vortexing, and left to standat room temperature for 45 minutes. After this the media was aspiratedfrom the plates, the DNA was added to the cells using a sterile plasticpipette, and the cells were left at room temperature for 20 minutes. Atthe end of this period, 5 ml of complete α(+)MEM containing 10% FBS wasadded to the plates, which were incubated at 37° C. for 4-6 hours. Themedia was then aspirated off the plates, and the cells were subjected toa glycerol shock by incubating them with a solution of 15% glycerol intransfection buffer at 37° C. for 3.5 minutes. After removal of theglycerol solution, the cells were washed twice with PBS, refed with 10ml complete α(+)MEM, 10% FBS, and returned to the 37° C. incubator. Forstable transfections, after 48 hours the cells were split 1:10 and fedwith selection medium (complete α-minus MEM (lacking nucleosides), 10%dialyzed FBS, and 0.02 μM methotrexate). Non-transfected (non-resistant)cells were typically eliminated in 3-4 weeks, leaving a population oftransfected, methotrexate-resistant cells.

3. Quantitation of Expression

Secretion of the hybrid proteins by transfected cells was assessed usinga commercial assay kit for soluble p55 (R&D Systems; Minneapolis, Minn.)in accordance with the manufacturer's instructions. This assay alsoprovides an estimate of the hybrid protein levels in conditioned andprocessed media, which served as the basis for selecting doses to beused in the bioassay.

4. Assessment of Heterodimer Formation

To assess the ability of the TBP-hCG subunit fusions to combine and formheterodimers, a sandwich immunoassay using antibodies to the hCGsubunits was performed. In this assay, a monoclonal antibody to the hCGβ subunit is coated onto microtiter plates and used for analyte capture.The primary detection antibody is a goat polyclonal raised against thehuman TSH α subunit (#082422G—Biodesign International; Kennenbunkport,Me.), which is in turn detected using a horse radish peroxidaseconjugated rabbit anti-goat polyclonal antibody (Cappel; Durham, N.C.).

Several different anti-hCG β subunit antibodies were used in this work,all of which show no detectable cross-reactivity with the free αsubunit. One of these antibodies (3/6) is used in the commerciallyavailable MAIAclone hCG assay kit (Biodata; Rome, Italy).

High-protein binding microtiter plates (Costar #3590) were coated withcapture antibody by incubation (2 hours at 37° C.) with 100 μl/well of a5 μg/ml solution of antibody in coating buffer (PBS, pH 7.4, 0.1 mMCa⁺⁺, 0.1 mM Mg⁺⁺). After washing once with wash solution (PBS, pH7.4+0.1 Tween 20) the plate is blocked by completely filling the wells(˜400 μl/well) with blocking solution (3% bovine serum albumin (BSA;fraction V—A-4503 Sigma) in PBS, pH 7.4) and incubating for one hour at37° C. or overnight at 4° C. The plate is then washed twice with washsolution, and the reference and experimental samples, diluted in diluent(5 mg/ml BSA in PBS, pH 7.4) to yield a 100 μl volume, are added. Afterincubating the samples and the plate for two hours at 37° C., the plateis again twice washed with wash solution. The primary detectionantibody, diluted 1:5000 in diluent, is added (100 μl/well) andincubated for one hour at 37° C. The secondary detection antibody (HRPconjugated rabbit anti-goat Ig), diluted 1:5000 in diluent, is added(100 μl/well) and after incubation for one hour at 37° C., the plate iswashed three times with wash solution. One hundred μl of TMB substratesolution (Kirkegaard and Perry Laboratories) is added, the plate isincubated 20 minutes in the dark at room temperature, and then theenzymatic reaction is stopped by addition of 50 μl/well 0.3M H₂SO₄. Theplate is then analyzed using a microtiter plate reader set for awavelength of 450 nm.

5. Partial Purification

To better quantitate the activities of these hybrid proteins, TBP-hCGhybrid proteins were partially purified by immunoaffinitychromatography. The antibody used was a monoclonal commerciallyavailable from R&D Systems (MAB #225). The column was CNBr-activatedsepharose, charged with the antibody by following the manufacturer's(Pharmacia) instructions.

Conditioned media was collected from confluent T-175 flasks of each lineusing daily harvests of 50 ml SFMII media (GIBCO), five harvests foreach line. The collections were subjected to centrifugation (1000 RPM)to remove cellular debris. The material was then assayed for TBP contentusing the commercial immunoassay and concentrated (Centricon units byAmicon; Beverly, Mass.) so that the apparent TBP concentration was about50 ng/ml.

Ten ml of the concentrated TBP-hCG (sample #18873) was brought toapproximately 1 M NaCl by addition of NaCl and adjustment of thesolution to a conductivity of approximately 85 mS/cm. This was passedthrough a 0.5 ml anti-TBP immunoaffinity column. The flow-through wascollected and run through the column a second time. After this thecolumn was washed with 1 M NaCl in PBS. The bound TBP(20-161)-hCG wascollected after elution with 50 mM citric acid (pH 2.5). The eluate(approximately 7 ml) was concentrated by filtration using AmiconCentricon-10's in accordance with the manufacturer's (Amicon)instructions, to a volume of approximately 200 μl. Approximately 800 μlof PBS was added to bring the sample volume to 1 ml, which was stored at4° C. until tested by bioassay.

6. Assessment of Anti-TNF Activity

Numerous in vitro TNF-induced cytotoxicity assays have been describedfor evaluating analogs of soluble TNF receptors. We utilized an assayemploying a human breast carcinoma cell line, BT-20 cells (ATCC HTB 19).The use of these cells as the basis for a TNF bioassay has beendescribed previously (48). These cells are cultured at 37° C. in RPMI1640 media supplemented with 10% heat-inactivated FBS. The cells weregrown to a maximum 80-90% confluence, which entailed splitting every 3-4days with a seeding density of about 3×10⁶ cells per T175 cm² flask.

The BT-20 assay uses the inclusion of a cellular stain, crystal violet,as a detection method to assess survival of cells after treatment withTNF. Dead cells are unable to take up and retain the dye.

In brief, the protocol used for the assay of anti-TNF activity is thefollowing. Recombinant human TNFα (R&D Systems) and the experimentalsamples are constituted in media (RPMI 1640 with 5% heat-inactivatedFBS) and added to the wells of 96-well culture plates. The cells arethen plated into these wells at a density of 1×10⁵ cells/well. Thequantity of TNFα added was determined earlier in titration studies, andrepresents a dose at which about 50% of the cells are killed.

After addition of the samples, the cells are cultured for 48 hours at39° C., after which the proportion of live cells is determined usingcrystal violet staining and a microtiter plate reader (570 nm).

RESULTS

1. Constructs Under Study

The designs of the hybrid proteins studied are briefly summarized below;two control proteins, a monomeric soluble p55 (r-hTBP-1) and a dimericTBP-immunoglobulin fusion protein (TBP-IgG3) (prepared essentially asdescribed in (10)), were studied for comparative purposes.

Fusion Construct TBP N-term TBP C-term partner r-hTBP-1 mix of 9 and 20180 none TBP-IgG3 mix of 9 and 20 190 IgG3 heavy chain constant regionTBP (20–161)-hCG 20 161 hCGα and hCGβ (heterodimer) TBP (20–190)-hCG 20190 hCGα and hCGβ (heterodimer)

The sequences of the DNAs encoding, TBP(20-190)-hCG and TBP(20-161)-hCGare provided in FIGS. 1 and 2, respectively. A schematic summary of theconstructs is provided in FIG. 3.

2. Secretion of TBP-hCG Proteins

All of the constructs tested were found to be produced and secreted intoculture media by transfected mammalian cells. Data illustrating this areshown in Tables 1 and 2.

TABLE 1 COS-7 transient expression (TBP ELISA) Concentration HybridProtein (pg/ml) TBP1 66 TBP-hCGα(20-161) 5.1 TBP-hCGβ(20-161) 0.5TBP-hCG(20-161) 2.7 Control <0.25

TABLE 2 COS-7 transient expression (TBP ELISA) Concentration Hybridprotein (ng/ml) TBP1 131 TBP-hCGα(20-190) 81 TBP-hCGβ(20-190) 9TBP-hCG(20-190) 62 Control <13. TBP-hCG(α/β) Fusion Proteins Assemble into Heterodimers

The combination of TBP-hCGα and TBP-hCGβ was confirmed using thesandwich assay for the hCG heterodimer. Only the combined transfectionof α and β subunit fusions resulted in heterodimer detection (Table 3).

TABLE 3 COS-7 transient expression (hCG heterodimer assay) ConcentrationHybrid Protein (ng/ml) TBP1 <0.2 TBP-hCGα(20-190) <0.2 TBP-hCGβ(20-190)<0.2 TBP-hCG(20-190) 38 Control <0.24. TBP-hCG Hybrid Proteins Exhibit Increased Activity Over TBP Monomer

Hybrid proteins produced in either COS-7 or CHO cells were found to bepotent inhibitors of TNFα in the BT-20 bioassay. Some of the samplestested are summarized in Table 4.

TABLE 4 Samples tested for anti-TNF activity Cell Construct sourceNature of sample r-hTBP-1 CHO purified TBP-IgG3 CHO 1x conditioned mediaTBP(20-161)-hCG CHO immunopurified (anti-TBP) TBP(20-190)-hCG CHO 1xconditioned media TBP(20-190)-hCG COS 1x conditioned media

Negative controls (conditioned media from mock transfections) wereincluded for the 1× media samples.

As illustrated in FIGS. 4-6 (points on y-axis), addition of TNF (2.5ng/ml) results in a clear reduction in live cell number (as assessed byOD 570). In every case, active samples have as a maximal protectiveeffect the restoration of cell viability to the level seen in theabsence of added TNF (i.e., the control labeled “cells alone”).

The positive controls, r-hTBP-1 and TBP-IgG3, are both protective,showing a clear dose-dependence and ED50s of approximately 100 ng/ml forthe r-hTBP-1 (FIGS. 4-6) and about 1.5 ng/ml for TBP-IgG3 (FIG. 4)respectively.

The TBP-hCG constructs from 1× media (CHO or COS) or from theimmunopurification show dose-dependent protection, with approximateED50s ranging from 2-11 ng/ml (FIGS. 4-6).

The results from the in vitro bioassay are reported in Table 5. The dataindicate that the hybrid proteins inhibit TNF cytotoxicity, and thatthey are substantially more potent than the TBP monomer. The negativecontrols were devoid of protective activity.

TABLE 5 Preliminary Assessment of the hybrid proteins in TNFCytotoxicity Assay Anti-TNF activity (ED50) in BT-20 Construct Fusionpartner bioassay** r-hTBP-1 none   100 ng/ml TBP-IgG3 IgG3 heavy chainconstant region  1.5 ng/ml TBP(20-161)-hCG hCGα and hCGβ (heterodimer)   2 ng/ml TBP(20-190)-hCG hCGα and hCGβ (heterodimer)  8-11 ng/ml **Thequantitation of material for dosing and estimation of ED50 was madeusing the TBP ELISA.

In addition to the possibility that dimerization of TBP may increasepotency, it is also possible that the activity of the hybrid proteinsare not related to dimeric interaction with TBP, but rather to stericinhibition due to the partner of the hybrid interfering with solubleTBP/TNF binding to cell-surface TNF receptors.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

Reference to known method steps, conventional method steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

REFERENCES

1. Smith, R. A. et al., J. Biol. Chem. 262:6951-6954, 1987.

2. Eck, M. J. et al., J. Biol. Chem. 264:17595-17605, 1989.

3. Jones, E. Y. et al , Nature 338:225-228, 1989.

4. Eck, M. J. et al., J. Biol. Chem. 267:2119-2122, 1992.

5. Pierce, J. G. et al., Annu. Rev. Biochem. 50:465-495, 1981.

6. Lapthorn, A. J. et al., Nature 369:455-461, 1994.

7. Wu, H., et al., Structure 2:545-550, 1994.

-   8. Engelmann, H., et al., J. Biol. Chem. 265:14497-14504, 1990.

9. Adam, D. et al., J. Biol. Chem. 270:17482-17487, 1995.

10. Loetscher, H. R., et al., J. Biol. Chem. 266:18324-18329, 1991.

11. Banner, D. W., et al., Cell 73:431-445, 1993.

12. Pennica, D., et al., Biochemistry 32:3131-3138, 1993.

13. Engelmann, H. et al., J. Biol. Chem. 265:1531-1536, 1990.

14. Van Zee, K. J. et al., Proc. Natl. Acad. Sci. USA 89:4845-4849,1992.

15. Aderka, D. et al., J. Exp. Med. 175:323-329, 1992.

16. Mohler, K. M., et al., J. Immunol. 151:1548-1561, 1993.

17. Bertini, R., et al., Eur. Cytokine Netw., 1993.

18. Piguet, P. F., et al., Immunology 77:510-514, 1992.

19. Williams, R. O., et al., Immunology 84:433-439, 1995.

20. Capon, D. J., et al., Nature 337: 525-531, 1989.

21. Ashkenazi, A., et al., Proc. Natl. Acad. Sci. 88:10535-10539, 1991.

22. Suitters, A. J., et al. J. Exp. Med. 179:849-856, 1994.

23. Nolan, O. et al., Biochim. Biophys. Acta 1040:1-11, 1990.

24. Rodrigues, M. L., et al., J. Immunol. 151:6954-6961, 1993.

25. Chang, H.-C., et al., Proc. Natl. Acad. Sci. USA 91:11408-11412,1994.

26. Wu, Z., et al., J. Biol. Chem. 270:16039-16044, 1995.

27. Bazzoni, F. et al, Proc. Natl. Acad. Sci. USA 92:5376-5380, 1995.

28. Boldin, M. P., et al., J. Biol. Chem. 270:387-391,1995.

29. Vu, T.-K. H., et al., Cell, 64:1057-1068, 1991.

30. Song, H. Y., et al., J. Biol. Chem. 269:22492-22495, 1994.

31. Russell, D. A., et al., J. Infectious Diseases 171:1528-1538, 1995.

32. Rao C. V. et al., Am. J. Obstet. Gynecol., 146, 65-68, 1983.

33. Damewood M. D. et al., Fertil. Steril. 52, 398-400, 1989.

34. Chen, F., et al., Mol. Endocrinol. 6:914-919, 1992.

35. Bielinska, M., et al., J. Cell Biol. 111:330a, 1990.

36. Furuhashi, M., et al., Mol Endocrinol. 9:54-63, 1995.

37. Sugahara, T., et al., Proc. Natl. Acad. Sci. USA 92:2041-2045, 1995.

38. Johnson, G. A., et al., Biol. Reprod. 52:68-73, 1995.

39. Urlaub, G. and Chasin, L. Proc. Natl. Acad. Sci. USA 77:4216-4220,1980.

40. Nophar, Y., et al., EMBO J. 9:3269-3278, 1990.

41. Fiddes, J. C. et al., Nature 281:351-356, 1979.

42. Fiddes, J. C. et al., Nature 286:684-687, 1980.

43. Elion, E. A., in Current Protocols in Molecular Biology, eds.Ausuble, F M. et al., John Wiley & Sons, 1993.

44. Campbell, R., Proc. Natl. Acad. Sci. USA 88:760-764, 1991.

45. Cole E. S. et al., Biotechnology, 11, 1014-1024, 1993.

46. Gluzman, Y., Cell 23:175-182, 1981.

47. Chu, G. et al., Nucl. Acid Res. 15:1311-1326, 1987.

48. Yen, J. et al., J. Immunotherapy 10:174-181, 1991.

1. A hybrid protein comprising two different coexpressed amino acidsequences forming a heterodimer, each comprising: (a) at least one aminoacid sequence selected from the group consisting of a chain of ahomomeric-receptor, a chain of a heteromeric receptor, a ligand otherthan a gonadotropin, a fragment of said chain of said homomericreceptor, said chain of said heteromeric receptor, or said ligand,wherein said ligand or fragment thereof retains ligand-receptor bindingcapability and said chain of said homomeric receptor or fragmentthereof, and said chain of said heteromeric receptor or fragment thereofretain ligand-receptor binding capability either alone or in associationwith a homologous or heterologous chain of said receptor; and (b) aheterodimeric scaffold which is a subunit of a circulatingnon-immunoglobulin protein, or a fragment thereof which retains theability of the subunit to form a heterodimer with other subunitsthereof; wherein sequences (a) and (b) are joined either directly orthrough a peptide linker, and in which the sequences (b) in each of saidtwo coexpressed sequences aggregate with each other to dimerize and forma heterodimer.
 2. A hybrid protein in accordance with claim 1, whereinsaid sequence (a) is selected from the group consisting of TNF BindingProtein 1 (TBP1), TNF Binding Protein 2 (TBP2) or a fragment of saidTBP1 or TBP2 still containing the ligand binding domain; theextracellular domain of the IFNα/β receptor or the IFNγ receptor; agonadotropin receptor or extracellular fragments thereof; and IL-6,IFNβ, thromhopoietin (TPO) or fragments thereof.
 3. A hybrid protein inaccordance with claim 1, wherein sequence (a) is joined, either directlyor via a linker, to the amino terminus of sequence (b).
 4. A hybridprotein in accordance with claim 1, wherein sequence (a) is joined,either directly or via a linker, to the carboxy terminus of sequence(b).
 5. A hybrid protein in accordance with claim 1, wherein said twocoexpressed amino acid sequences each include the sequence for TBP1 or afragment thereof having amino acid residues 20-161 or 20-190 of TBP1, assequence (a), wherein said two coexpressed amino acid sequences form aheterodimer through sequence (b).
 6. A hybrid protein in accordance withclaim 1, wherein said two coexpressed amino acid sequences each includethe extracellular domain of a gonadotropin receptor as sequence (a),wherein said two coexpressed amino acid sequences, form a heterodimerthrough sequence (b).
 7. A hybrid protein in accordance with claim 1,wherein said sequence (a) is the FSH receptor extracellular domain.
 8. Ahybrid protein in accordance with claim 6, wherein said sequences (a)and (b) are linked with a peptide linker.
 9. A hybrid protein inaccordance with claim 8, wherein said peptide linker has an enzymecleavage site.
 10. A hybrid protein in accordance with claim 9, whereinsaid enzyme cleavage site is a thrombin cleavage site.
 11. A hybridprotein in accordance with claim 9, wherein said enzyme cleavage site isrecognized and cleaved by an enzyme which is found in the ovary.
 12. Ahybrid protein in accordance with claim 8, wherein said peptide linkerserves as a flexible hinge.
 13. A pharmaceutical composition comprisinga hybrid protein in accordance with claim 1 and a pharmaceuticallyacceptable carrier and/or excipient.
 14. The hybrid protein of claim 1,wherein each of said two coexpressed amino acid sequences forming aheterodimer consists essentially of sequences (a) and (b).
 15. A methodfor inducing follicular maturation, comprising administering to asubject in need thereof a hybrid protein comprising two coexpressedamino acid sequences forming a dimer, each comprising FSH receptorextracellular domain and a subunit of FSH, wherein the FSH receptorextracellular domain and the subunit of FSH are bonded together directlyor through a peptide linker, and in which each FSH subunit in each ofsaid coexpressed sequences is capable of aggregating with the other FSHsubunit to form a dimer complex.
 16. The method of claim 15, wherein theFSH receptor extracellular domain is linked to the amino terminus of theFSH subunit.
 17. The method of claim 15, wherein the FSH receptorextracellular domain and the subunit of FSH are linked through a peptidelinker.
 18. The method of claim 17, wherein the peptide linker has anenzyme cleavage site.
 19. The method of claim 18, wherein the enzymecleavage site is a thrombin cleavage site.
 20. The method of claim 17,wherein the peptide linker serves as a flexible hinge.